Aromatic-aromatic interactions and protein stability. Investigation by double-mutant cycles

The side-chains of phenylalanine and tyrosine residues in proteins are frequently found to be involved in pairwise interactions. These occur both within repeating elements of secondary structure and in tertiary and quaternary interactions. It has been suggested that they are important in protein folding and stability, and non-bonded potential energy calculations indicate that a typical aromatic-aromatic interaction has an energy of between -1 and -2 kcal/mol and contributes between -0.6 and -1.3 kcal/mol to protein stability. There is such an aromatic pair on the solvent-exposed face of the first alpha-helix of barnase, the small ribonuclease from Bacillus amyloliquefaciens. The edge of the aromatic ring of Tyr17 interacts with the face of that of Tyr13. The two residues have been mutated both singly and pairwise to alanine, and their free energies of unfolding determined by denaturation with urea. Application of the double-mutant cycle analysis gives an interaction energy of -1.3 kcal/mol for the aromatic pair in the folded protein relative to solvation by water in the unfolded protein. This value is similar to that calculated from the change in surface-accessible area between the rings on the formation of the pair. Analysis of a further double-mutant cycle in which the Tyr residues are mutated to Phe indicates that the aromatic-aromatic interactions of Tyr/Tyr and Phe/Phe make identical contributions to protein stability. However, Tyr is preferred to Phe by 0.3(+/- 0.04) kcal/mol at the solvent-exposed face of the alpha-helix.     

Inter-residue and solvent-residue interactions in proteins: a statistical study on experimental structures

A large set of protein structures resolved by X-ray or NMR techniques has been extracted from the Protein Data Bank and analyzed using statistical methods. In particular, we investigate the interactions between side chains and the interactions between solvent and side chains, pointing out on the possibility of including the solvent as part of a knowledge-based potential. The solvent-residue contacts are accounted for on the basis of the Voronoi's polyhedron analysis. Our investigation confirms the importance of hydrophobic residues in determining the protein stability. We observe that in general hydrophobic-hydrophobic interactions and, more specifically, aromatic-aromatic contacts tend to be increasingly distally separated in the primary sequence of proteins, thus connecting distinct secondary structure elements. A simple relation expressing the dependence of the protein free energy by the number of residues is proposed. Such a relation includes both the residue-residue and the solvent-residue contributions. The former is dominant for large size proteins, whereas for small sizes (number of residues less than 100) the two terms are comparable. Gapless threading experiments show that the solvent-residue knowledge-based potential yields a significant contribution with respect to discriminating the native structure of proteins. Such contribution is important especially for proteins of small size and is similar to that given by the most favorable residue-residue knowledge-based potential referring to hydrophobic-hydrophobic interactions such as isoleucine-leucine. In general, the inclusion of the solvent-residue interaction produces a relevant increase of the free energy gap between the native structures and decoys.     

Aromatic-aromatic interactions in the formation of the MDM2-p53 complex

Although the molecular interaction of MDM2 with the transactivation domain of p53 has been thoroughly studied, there is very limited information regarding the steps involved in the recognition mechanism between these proteins. On this basis, we performed four high-temperature molecular dynamics simulations in explicit solvent to gain insight into the interactions involved in the fist contact toward the formation of the complex. We found that the presence of specific intermolecular aromatic pairs at the interface of the complex, around the native-like state of MDM2, is consistent among independent molecular dynamics runs. This observation suggests that aromatic-aromatic interactions are closely related to the first contact between MDM2 and p53. Thus, we propose that aromatic-aromatic interactions are an important, and probably essential, requirement for the formation and stabilization of the MDM2-p53 complex.     

Amino-aromatic interactions in proteins

Geometric analysis of 33 refined high-resolution protein crystal structures (2 A or higher) demonstrates that side-chain amino groups interact with aromatic side chains. Positively charged or delta(+) amino groups of lysine, arginine, asparagine, glutamine and histidine are preferentially located within 6 A of the ring centroids of phenylalanine, tyrosine and tryptophan, where they make van der Waals' contact with the delta(-) pi-electrons and avoid the delta(+) ring edge. This geometric pattern is different from the distribution expected due to random close packing of side chains in a protein. It is opposite to oxygen- and sulfur-aromatic interactions, similar to aromatic-aromatic interactions, and almost certainly electrostatic in origin.     

Recognition schemes for protein-nucleic acid interactions

The molecular forces involved in protein-nucleic acid interaction are electrostatic, stacking and hydrogen-bonding. These interactions have a certain amount of specificity due to the directional nature of such interactions and the spatial contributions of the steric effects of different substituent groups. Quantum chemical calculations on these interactions have been reported which clearly bring out such features. While the binding energies for electrostatic interactions are an order of magnitude higher, the differences in interaction energies for structures stabilised by hydrogen-bonding and stacking are relatively small. Thus, the molecular interactions alone cannot explain the highly specific nature of binding observed in certain segments of proteins and nucleic acids. It is therefore logical to assume that the sequence dependent three dimensional structures of these molecules help to place the functional groups in the correct geometry for a favourable interaction between the two molecules. We have carried out 2D-FT nuclear magnetic resonance studies on the oligonucleotide d-GGATCCGGATCC. This oligonucleotide sequence has two binding sites for the restriction enzyme Bam H1. Our studies indicate that the conformation of this DNA fragment is predominantly B-type except near the binding sites where the ribose ring prefers a³E conformation. This interesting finding raises the general question about the presence of specificity in the inherent backbone structures of proteins and nucleic acids as opposed to specific intermolecular interactions which may induce conformational changes to facilitate such binding.     

Aromatic and cation-pi interactions enhance helix-helix association in a membrane environment

The cation-pi interaction is an electrostatic attraction between a positive charge and the conjugated pi electrons of an aromatic ring. These interactions are well documented in soluble proteins and can be both structurally and functionally important. Catalyzed by observations in our laboratory that an Ala- and Ile-rich two-helix transmembrane segment tended to form SDS-resistant dimers upon the incorporation of suitably located Trp residues, here we have constructed a library of related constructs to study systematically the impact of aromatic-aromatic and cation-pi interactions on tertiary structure formation within an Escherichia coli membrane. Using the TOXCAT oligomerization assay with the hydrophobic segment AIAIAIIAZAXAIIAIAIAI, where Z = A, W, Y, or F and X = A, H, R, or K in all possible combinations of cation and/or aromatic pairings, to assess the TM-TM dependent expression of the chloramphenicol acetyltransferase reporter gene, we find that cation-pi interactions, particularly between Lys and Trp, Tyr, or Phe, as well as weakly polar interactions between pairs of aromatic residues, significantly enhance the strength of oligomerization of these hydrophobic helices, in some instances forming oligomers four times stronger than the high-affinity glycophorin A dimer. The contribution of these forces to the tertiary structure formation in designed transmembrane segments suggests that similar forces may also be a significant factor in the folding and stability of native membrane proteins.     

Aromatic-aromatic interactions in the zinc finger motif. Analysis of the two-dimensional nuclear magnetic resonance structure of a mutant domain.

The folding and stability of globular proteins are determined by a variety of chemical mechanisms, including hydrogen bonds, salt bridges and the hydrophobic effect. Of particular interest are weakly polar interactions involving aromatic rings, which are proposed to regulate the geometry of closely packed protein interiors. Such interactions reflect the electrostatic contribution of pi-electrons and, unlike van der Waals' interactions and the hydrophobic effect, may, in principle, introduce a directional force in a protein's hydrophobic core. Although the weakly polar hypothesis is supported by a statistical analysis of protein structures, the general importance of such contributions to protein folding and stability is unclear. Here, we show the presence of alternative aromatic-aromatic interactions in the two-dimensional nuclear magnetic resonance structure of a mutant Zn finger. Changes in aromatic packing lead in turn to local and non-local differences between the structures of a wild-type and mutant domain. The results provide insight into the evolution of Zn finger sequences and have implications for understanding how geometric relationships may be chemically encoded in a simple sequence template.     

Optimization of the electrostatic interactions in proteins of different functional and folding type

The 3-dimensional optimization of the electrostatic interactions between the charged amino acid residues was studied by Monte Carlo simulations on an extended representative set of 141 protein structures with known atomic coordinates. The proteins were classified by different functional and structural criteria, and the optimization of the electrostatic interactions was analyzed. The optimization parameters were obtained by comparison of the contribution of charge-charge interactions to the free energy of the native protein structures and for a large number of randomly distributed charge constellations obtained by the Monte Carlo technique. On the basis of the results obtained, one can conclude that the charge-charge interactions are better optimized in the enzymes than in the proteins without enzymatic functions. Proteins that belong to the mixed αβ folding type are electrostatically better optimized than pure α-helical or β-strand structures. Proteins that are stabilized by disulfide bonds show a lower degree of electrostatic optimization. The electrostatic interactions in a native protein are effectively optimized by rejection of the conformers that lead to repulsive charge-charge interactions. Particularly, the rejection of the repulsive contacts seems to be a major goal in the protein folding process. The dependence of the optimization parameters on the choice of the potential function was tested. The majority of the potential functions gave practically identical results.     

Functional interactions in bacteriorhodopsin: a theoretical analysis of retinal hydrogen bonding with water.

The light-driven proton pump, bacteriorhodopsin (bR) contains a retinal molecule with a Schiff base moiety that can participate in hydrogen-bonding interactions in an internal, water-containing channel. Here we combine quantum chemistry and molecular mechanics techniques to determine the geometries and energetics of retinal Schiff base-water interactions. Ab initio molecular orbital calculations are used to determine potential surfaces for water-Schiff base hydrogen-bonding and to characterize the energetics of rotation of the C-C single bond distal and adjacent to the Schiff base NH group. The ab initio results are combined with semiempirical quantum chemistry calculations to produce a data set used for the parameterization of a molecular mechanics energy function for retinal. Using the molecular mechanics force field the hydrated retinal and associated bR protein environment are energy-minimized and the resulting geometries examined. Two distinct sites are found in which water molecules can have hydrogen-bonding interactions with the Schiff base: one near the NH group of the Schiff base in a polar region directed towards the extracellular side, and the other near a retinal CH group in a relatively nonpolar region, directed towards the cytoplasmic side.     

Monte Carlo simulation of protein-induced lipid demixing in a membrane with interactions derived from experiment

Lipid domain formation induced by annexin was investigated in mixtures of phosphatidylcholine (PC), phosphatidylserine (PS), and cholesterol (Chol), which were selected to mimic the inner leaflet of a eukaryotic plasma membrane. Annexins are ubiquitous and abundant cytoplasmic, peripheral proteins, which bind to membranes containing PS in the presence of calcium ions (Ca²⁺), but whose function is unknown. Prompted by indications of interplay between the presence of cholesterol in PS/PC mixtures and the binding of annexins, we used Monte Carlo simulations to investigate protein and lipid domain formation in these mixtures. The set of interaction parameters between lipids and proteins was assigned by matching experimental observables to corresponding variables in the calculations. In the case of monounsaturated phospholipids, the PS-PC and PC-Chol interactions are weakly repulsive. The interaction between protein and PS was determined based on experiments of annexin binding to PC/PS mixtures in the presence of Ca²⁺. Based on the proposal that PS and cholesterol form a complex in model membranes, a favorable PS-Chol interaction was postulated. Finally, protein-protein favorable interactions were also included, which are consistent with observations of large, two-dimensional, regular arrays of annexins on membranes. Those net interactions between pairs of lipids, proteins and lipids, and between proteins are all small, of the order of the average kinetic energy. We found that annexin a5 can induce formation of large PS domains, coincident with protein domains, but only if cholesterol is present.     

On the calculation of electrostatic interactions in proteins

In this paper we present a classical treatment of electrostatic interactions in proteins. The protein is treated as a region of low dielectric constant with spherical charges embedded within it, surrounded by an aqueous solvent of high dielectric constant, which may contain a simple electrolyte. The complete analysis includes the effects of solvent screening, polarization forces, and self energies, which are related to solvation energies. Formulae, and sample calculations of forces and energies, are given for the special case of a spherical protein. Our analysis and model calculations point out that any consistent treatment of electrostatic interactions in proteins should account for the following. Solvent polarization is an important factor in the calculation of pairwise electrostatic interactions. Solvent polarization substantially affects both electrostatic energies and forces acting upon charges. No simple expression for the effective dielectric constant, Deff, can generally be valid, since Deff is a sensitive function of position. Solvent screening of pairwise interactions involving dipolar groups is less effective than the screening of charges. In fact for many interactions involving dipoles, solvent screening can be essentially ignored. The self energy of charges makes a large contribution to the total electrostatic energy of a protein. This must be compensated by specific interactions with other groups in the protein. Strategies for applying our analysis to proteins whose structures are known are discussed.     

Aromatic-aromatic interactions in and around alpha-helices

To understand the role of aromatic-aromatic interactions in imparting specificity to the folding process, the geometries of four aromatic residues with different sequence spacing, located in alpha-helices or five residues from helical ends, interacting with each other have been elucidated. The geometry is found to depend on the sequence difference. Specific interactions (C-H...pi and N-H...pi) which result from this geometry may cause a given pair of residues (such as Phe-His) with a particular sequence difference to occur more than expected. The most conspicuous residue in an aromatic pair in the context of helix stability is His, which is found at the last (C1) position or the two positions (Ncap and Ccap) immediately flanking the helix. An alpha-helix and a contiguous 3(10)-helix or two helices separated by a non-helical residue can have interacting aromatic pairs, the geometry of interaction and the relative orientation between the helices being rather fixed. Short helices can also have interacting residues from either side.     

Cation-pi (Na+-Trp) interactions in the crystal structure of tetragonal lysozyme.

Experimental evidence of a cation-pi interaction between a sodium cation (Na+) and the indole ring of residue Trp123 in a structure (2.0 A) of hen egg-white lysozyme is presented. The geometry of the metal ion-pi interaction observed in the protein structure (distance between the aromatic plane and the cation approximately 4 A) is consistent with geometries observed among small molecules crystal structures and quantum chemistry ab initio calculations. The present crystal structure of lysozyme provides unique structural information about the geometry of binding of cations to pi systems in proteins. It shows that the metal ion-pi interaction within proteins is not significantly different from similar bindings found in small molecules and that it can be modeled by theoretical methods.     

Molecular simulations of lipid-mediated protein-protein interactions

Recent experimental results revealed that lipid-mediated interactions due to hydrophobic forces may be important in determining the protein topology after insertion in the membrane, in regulating the protein activity, in protein aggregation and in signal transduction. To gain insight into the lipid-mediated interactions between two intrinsic membrane proteins, we developed a mesoscopic model of a lipid bilayer with embedded proteins, which we studied with dissipative particle dynamics. Our calculations of the potential of mean force between transmembrane proteins show that hydrophobic forces drive long-range protein-protein interactions and that the nature of these interactions depends on the length of the protein hydrophobic segment, on the three-dimensional structure of the protein and on the properties of the lipid bilayer. To understand the nature of the computed potentials of mean force, the concept of hydrophilic shielding is introduced. The observed protein interactions are interpreted as resulting from the dynamic reorganization of the system to maintain an optimal hydrophilic shielding of the protein and lipid hydrophobic parts, within the constraint of the flexibility of the components. Our results could lead to a better understanding of several membrane processes in which protein interactions are involved.     

The optimization of protein-solvent interactions: thermostability and the role of hydrophobic and electrostatic interactions.

Protein-solvent interactions were analyzed using an optimization parameter based on the ratio of the solvent-accessible area in the native and the unfolded protein structure. The calculations were performed for a set of 183 nonhomologous proteins with known three-dimensional structure available in the Protein Data Bank. The dependence of the total solvent-accessible surface area on the protein molecular mass was analyzed. It was shown that there is no difference between the monomeric and oligomeric proteins with respect to the solvent-accessible area. The results also suggested that for proteins with molecular mass above some critical mass, which is about 28 kDa, a formation of domain structure or subunit aggregation into oligomers is preferred rather than a further enlargement of a single domain structure. An analysis of the optimization of both protein-solvent and charge-charge interactions was performed for 14 proteins from thermophilic organisms. The comparison of the optimization parameters calculated for proteins from thermophiles and mesophiles showed that the former are generally characterized by a high degree of optimization of the hydrophobic interactions or, in cases where the optimization of the hydrophobic interactions is not sufficiently high, by highly optimized charge-charge interactions.     

Stereochemistry of guanidine-metal interactions: implications for L-arginine-metal interactions in protein structure and function

The geometries of 150 guanidine-metal ion interactions retrieved from crystal structures deposited in the Cambridge Structural Database have been analyzed. Metal ions exhibit a preference for anti coordination stereochemistry in the plane of the unprotonated guanidine group, usually in chelate complexes with a diguanidine moiety, but syn-oriented interactions are occasionally found for single guanidine-metal interactions. Three L-arginine-metal coordination interactions are found in metalloenzyme structures deposited in the Protein Data Bank: biotin synthase from E. coli, His-67 --> Arg human carbonic anhydrase I, and inactivated B. caldovelox arginase complexed with L-arginine. In these proteins, L-arginine-metal coordination adopts syn/out-of-plane and anti/in-plane coordination stereochemistry. The implications of these results for L-arginine-metal interactions in protein structure and function are discussed. Although such interactions are rare, this analysis serves as a useful reference point for the growing interest in enzymes containing L-arginine residues that function as general bases or metal ligands.     

Molecular simulation of the effect of cholesterol on lipid-mediated protein-protein interactions

Experiments and molecular simulations have shown that the hydrophobic mismatch between proteins and membranes contributes significantly to lipid-mediated protein-protein interactions. In this article, we discuss the effect of cholesterol on lipid-mediated protein-protein interactions as function of hydrophobic mismatch, protein diameter and protein cluster size, lipid tail length, and temperature. To do so, we study a mesoscopic model of a hydrated bilayer containing lipids and cholesterol in which proteins are embedded, with a hybrid dissipative particle dynamics-Monte Carlo method. We propose a mechanism by which cholesterol affects protein interactions: protein-induced, cholesterol-enriched, or cholesterol-depleted lipid shells surrounding the proteins affect the lipid-mediated protein-protein interactions. Our calculations of the potential of mean force between proteins and protein clusters show that the addition of cholesterol dramatically reduces repulsive lipid-mediated interactions between proteins (protein clusters) with positive mismatch, but does not affect attractive interactions between proteins with negative mismatch. Cholesterol has only a modest effect on the repulsive interactions between proteins with different mismatch.     

Thermodynamic penalty arising from burial of a ligand polar group within a hydrophobic pocket of a protein receptor

Here, we examine the thermodynamic penalty arising from burial of a polar group in a hydrophobic pocket that forms part of the binding-site of the major urinary protein (MUP-I). X-ray crystal structures of the complexes of octanol, nonanol and 1,8 octan-diol indicate that these ligands bind with similar orientations in the binding pocket. Each complex is characterised by a bridging water molecule between the hydroxyl group of Tyr120 and the hydroxyl group of each ligand. The additional hydroxyl group of 1,8 octan-diol is thereby forced to reside in a hydrophobic pocket, and isothermal titration calorimetry experiments indicate that this is accompanied by a standard free energy penalty of +21 kJ/mol with respect to octanol and +18 kJ/mol with respect to nonanol. Consideration of the solvation thermodynamics of each ligand enables the "intrinsic" (solute-solute) interaction energy to be determined, which indicates a favourable enthalpic component and an entropic component that is small or zero. These data indicate that the thermodynamic penalty to binding derived from the unfavourable desolvation of 1,8 octan-diol is partially offset by a favourable intrinsic contribution. Quantum chemical calculations suggest that this latter contribution derives from favourable solute-solute dispersion interactions.     

Het-PDB Navi.: a database for protein-small molecule interactions

The genomes of more than 100 species have been sequenced, and the biological functions of encoded proteins are now actively being researched. Protein function is based on interactions between proteins and other molecules. One approach to assuming protein function based on genomic sequence is to predict interactions between an encoded protein and other molecules. As a data source for such predictions, knowledge regarding known protein-small molecule interactions needs to be compiled. We have, therefore, surveyed interactions between proteins and other molecules in Protein Data Bank (PDB), the protein three-dimensional (3D) structure database. Among 20,685 entries in PDB (April, 2003), 4,189 types of small molecules were found to interact with proteins. Biologically relevant small molecules most often found in PDB were metal ions, such as calcium, zinc, and magnesium. Sugars and nucleotides were the next most common. These molecules are known to act as cofactors for enzymes and/or stabilizers of proteins. In each case of interactions between a protein and small molecule, we found preferred amino acid residues at the interaction sites. These preferences can be the basis for predicting protein function from genomic sequence and protein 3D structures. The data pertaining to these small molecules were collected in a database named Het-PDB Navi., which is freely available at http://daisy.nagahama-i-bio.ac.jp/golab/hetpdbnavi.html and linked to the official PDB home page.